The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+AML cell lines KG1a, Kasumi-1, DNR-sensitive U937 AML cells, and primary CD34+AML
Trang 1R E S E A R C H Open Access
Curcumin reduces expression of Bcl-2, leading to
acute myeloid leukemia cell lines and primary
Jia Rao1,2, Duo-Rong Xu2,3*, Fei-Meng Zheng4, Zi-Jie Long1,2, Sheng-Shan Huang5, Xing Wu1,2, Wei-Hua Zhou1,2, Ren-Wei Huang1,2*and Quentin Liu1,2,4*
Abstract
Background: Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis CD34+AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34-AML cells Curcumin is a major component of turmeric that has shown cytotoxic activity in
multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+AML cells The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+AML bone-marrow-derived cells
Methods: Primary human CD34+cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays Cell cycle distribution was examined by propidium iodide (PI) assay Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays The change in mitochondrial
membrane potential (MMP) was examined by JC-1 staining and flow cytometry Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting Short interfering RNA (siRNA) against Bcl-2 was used in CD34+KG1a and Kasumi-1 cells incubated with/without DNR
Results: Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells,
consistent with decreased Bcl-2 expression Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis More importantly, curcumin suppressed Bcl-2 expression, selectively inhibited proliferation and synergistically enhanced the cytotoxicity of DNR in primary CD34+AML cells, while showing limited lethality in normal CD34+hematopoietic progenitors
Conclusion: Curcumin down-regulates Bcl-2 and induces apoptosis in DNR-insensitive CD34+AML cell lines and primary CD34+AML cells
* Correspondence: xudr@hotmail.com; huangrw56@163.com; liuq9@mail.sysu.
edu.cn
1
Department of Hematology, Third Affiliated Hospital, Sun Yat-sen University,
600 Tianhe Road, Guangzhou 510630, P.R China
2
Sun Yat-sen Institute of Hematology, 600 Tianhe Road, Guangzhou 510630,
P.R China
Full list of author information is available at the end of the article
© 2011 Rao et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
Trang 2Acute myeloid leukemia (AML) is an
immunophenotypi-cally heterogenous malignant disease, in which CD34
posi-tivity has been significantly correlated with a lower
complete response (CR) rate, drug resistance and poor
outcome [1-3] Treatment of AML has generally consisted
of a combination of cytarabine and an anthracycline such
as daunorubicin (DNR), or the anthracenedione
mitoxan-trone [4] Although conventional chemotherapy regimens
induce CR in 65-80% of newly diagnosed AML patients,
most patients who achieve a CR relapse within 2 years
from diagnosis [5] At relapse, blast cells usually display a
more immature phenotype, with one of the most common
antigenic changes being a gain in expression of the stem
cell antigen CD34 [6,7] This is reflected in the resistance
of these immature phenotype CD34+AML progenitors to
current chemotherapies
CD34+ AML cells are 10-15-fold more resistant to
DNR than CD34-AML cells [8] CD34+ KG1a and TF-1
AML cell lines are 30-40 fold more resistant to
mitox-antrone than more mature HL-60 and U937 cells, and
this resistance appears to be associated with the lack of
apoptosis [9] Increasing evidence indicates that CD34+
AML cells are less sensitive to spontaneous apoptosis
and have higher levels of Bcl-2 and Bcl-xl gene and
pro-tein expression than the CD34-subpopulation [6,10-12]
CD34 positivity has been reported to be another
indica-tor of poor prognosis in AML [3,12], and use of more
effective drugs to eliminate this early immature CD34+
AML cell subpopulation might therefore improve the
outcome of AML
DNR is one of the most commonly used anti-leukemia
agents Bcl-2 overexpression can block DNR-induced
apoptosis in more mature U937 AML cells [13] The
anti-apoptotic proteins Bcl-2 and Bcl-xl also contribute
to the survival and chemoresistance of quiescent
leuke-mia CD34+ cells [14] These findings suggest that Bcl-2
plays a critical role in CD34+AML cell survival and that
agents aimed at down-regulating Bcl-2 protein might be
effective for the treatment of DNR-insensitive CD34+
AML
Curcumin, a major yellow pigment in turmeric, has
been proven to be a powerful therapeutic drug [15,16]
Curcumin induces apoptosis in a variety of tumor cells,
including more mature HL-60 and U937 cell lines,
through activation of caspase-3, cytochrome c release,
and down-regulation of Bcl-2 [17-20] Curcumin inhibits
proliferation in a variety of cancer cells through
target-ing multiple cellular signaltarget-ing pathways [21], includtarget-ing
the mitogen-activated protein kinase [22], nuclear factor
kappaB [23], phosphoinositide-3 kinase/Akt/mammalian
target of rapamycin [24,25], Wnt [26], and
Notch-mediated signaling pathways [27] Curcumin has also
been found to be a powerful chemosensitizing agent in tumor cells It demonstrated no major toxicities in phase I and II clinical studies at doses of up to 8 g/day [28,29] However, the cytotoxic effects of curcumin in DNR-insensitive CD34+ immature AML cells remain unclear
In this study, we examined the cytotoxic efficiency and molecular mechanisms underlying the anticancer activity
of curcumin in both DNR-insensitive CD34+ immature AML cell lines and in primary CD34+AML cells
Methods
Materials Curcumin (Sigma, St Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100-mM stock solution that was stored at -20°C DNR was purchased from Pharmacia & Upjohn SpA (Milan, Italy) Annexin-V assay kit was purchased from Molecular Probes (Eugene,
OR, USA) Anti-cleaved PARP, cleaved caspase-3, and Bcl-2 antibodies were purchased from Cell Signaling Technologies (Beverly, MA, USA) Anti-GAPDH anti-body and goat anti-rabbit/mouse-horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Protein Tech Group (Chicago, IL, USA) JC-1 kit was purchased from Beyotime (China) CD34-PE and IgG1-PE monoclonal antibodies were purchased from BD Biosciences (San Jose, CA, USA) CD34 MicroBead kit was purchased from Miltenyi biotec (Auburn, CA, USA)
Cell lines, primary samples, and cell culture KG1a and Kasumi-1 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkul-turen GmbH (DSMZ) (Braunschweig, Germany) and grown in RPMI 1640 medium (Gibco; Invitrogen, Carls-bad, CA, USA) supplemented with 20% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT) According to immu-nological studies by DSMZ and others [30,31], KG1a and Kasumi-1 cells are characterized by high expression of CD34 surface antigen U937 cells were obtained from the American Type Culture Collection (ATCC) and grown in RPMI 1640 medium supplemented with 10% FBS Cells were cultured at 37°C in a humidified atmosphere con-taining 5% CO2 Control cultures received an equivalent amount of DMSO only Bone marrow mononuclear cells (BMMCs) or mobilized peripheral blood mononuclear cells (PBMCs) were obtained from 9 newly diagnosed AML patients and 8 healthy donors All donors provided written informed consent, and the study had the approval
of the Institute Research Ethics Committee at Sun Yan-sen University, in accordance with the Declaration of Helsinki Patient characteristics are shown in Table 1 PBMCs and BMMCs were enriched by Ficoll-Hypaque density gradient centrifugation and isolated using a CD34
Trang 3MicroBead kit BMMCs and PBMCs were stained with
PE-conjugated anti-CD34 to determine the purity of
CD34+cells
MTT assay
Viability was assessed by MTT assay Briefly, 1.0×104
cells were incubated in triplicate in a 96-well plate in the
presence or absence of the indicated test samples in a
final volume of 0.2 ml for various lengths of time at 37°C
Thereafter, 20μl MTT solution (5 mg/ml in PBS) was
then added to each well After 4-h incubation at 37°C,
150μl DMSO was added Finally the plates were shaken
and the optical density at 490 nm was measured using a
multiwell plate reader (Microplate Reader; Bio-Rad,
Hercules, CA) Percent cell viability was calculated as cell
viability of the experimental samples/cell viability of the
control samples × 100 At least three independent
experi-ments were performed
Colony-forming assay
Treated and untreated cells were cultured in RPMI 1640
medium supplemented with 0.9% methylcellulose and
20% FBS at 37°C in 5% CO2 The colonies (containing
50 or more cells) were counted by light microscopy
after 14 days All semi-solid cultures were performed in
triplicate Three independent experiments were
performed
Wright-Giemsa staining
Morphological signs of apoptosis were detected by
Wright-Giemsa staining Cells were treated with 0-80μM
curcumin for 24 h Smears of control and treated cells
were stained with Wright-Giemsa solution for 25 min,
rinsed with distilled water and air dried Cell morphology
was studied by light microscopy
Hoechst 33342 staining
Nuclear fragmentation was examined by Hoechst 33342
(Sigma) Cells treated with 0-80μM curcumin for 24 h
were washed and stained with Hoechst 33342 (10μg/ml) for 15 min at 37°C Slides were viewed using a fluores-cence microscope
Measurement of apoptosis by Annexin V analysis
An Annexin V-binding assay was used according to the manufacturer’s instructions Briefly, approximately 5×105/ml cells in 6-well plates were treated with various concentrations of the indicated test samples The cells were harvested and used for Annexin V-Alexa Fluor-488/PI staining The stained cells were analyzed by flow cytometry to determine the percentages of AnnexinV
+
/PI-(early apoptosis) and AnnexinV+/PI+ (late apopto-sis) cells
Cell cycle analysis Cell cycle was analyzed by flow cytometry Approxi-mately 5 × 105/ml cells in 6-well plates were treated with various concentrations of curcumin for 24 h Cell cycle analysis was performed using the CycleTEST™ PLUS DNA kit (BD Biosciences)
Detection of mitochondrial membrane potential (MMP, Δψm) using JC-1
MMP was estimated by flow cytometry after staining with JC-1 fluorescent dye When the cell is in a normal state, MMP is high and JC-1 predominantly appears as red fluorescence When the cell is in an apoptotic or necrotic state, the MMP is reduced and JC-1 appears as a monomer indicated by green fluorescence A change in the florescence from red to green indicates a decrease in the MMP Approximately 5×105/ml cells in 6-well plates were treated with various concentrations of curcumin for
24 h The cells were then washed with PBS and incubated with JC-1 working solution for 20 min at 37°C in the dark Cells were washed with PBS and resuspended in
500μl PBS The stained cells were analyzed by flow cyto-metry to determine the change in the florescence from red to green
Table 1 Characteristic of patients
P patient, Y year, M male, FAB French-American-British, WBC white blood cells, BMC bone marrow cells, BM bone marrow, PM peripheral blood.*Percentage of CD34 +
cells in bone marrow cells of AML patients before sorting #
The t (8; 21) (q22; q22) chromosomal translocation gives rise to the AML1/ETO fusion oncoprotein.
Trang 4RNA isolation and semiquantitative reverse
transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol isolation reagent
(Invitrogen, USA) Reverse transcription was performed
using a reverse transcriptase first strand cDNA synthesis
kit (Takara, Japan) The sequences of the sense and
anti-sense primers were: 5’-CTGGTGGACAACATCGC-3’
(sense) and 5’-GGAGAAATCAAACAGAGGC-3’
(anti-sense) for Bcl-2, 5’-TGACTTTTCCTGTGAACTCT-3’
(sense) and 5’-GCCTTTCATTCGTATCAAGA-3’
(anti-sense) for c-IAP-1, 5’-GCAGGGTTTCT TTATACTG-3’
(sense) and 5’-TGTCCCTTCTGTTCTAACAG-3’
(anti-sense) for XIAP [32],
5’-GTGGACATCCGCAAAGAC-3’ (sense) and 5’-GAAAGGGTGTAA CGCAACT-5’-GTGGACATCCGCAAAGAC-3’
(anti-sense) forb-actin The PCR conditions were as
fol-lows: for c-IAP-1 and XIAP, 94°C for 1 min, 62°C for
1 min, and 72°C for 1 min; for Bcl-2, 94°C for 30 s,
62°C for 30 s, 72°C for 10 s; and for b-actin: 94°C for
30 s, 55°C for 30 s, 72°C for 1 min Thirty cycles of
amplification were used PCR (10μl) products were
ana-lyzed by electrophoresis on 2% (w/v) agarose gel
Western blot analysis
Total cellular proteins were isolated with lysis buffer
(20 mM Tris, pH 7.5; 150 mM NaCl; 0.25% NP40;
2.5 mM sodium pyprophosphate; 1 mM EGTA, 1 mM
EDTA; 1 mMb-glycerophosphate; 1 mM Na3VO4; 1 mM
PMSF; 1μg/ml leupeptin) Equal amounts of protein were
subjected to 10% or 15% sodium dodecyl
sulfate-polyacry-lamide gel electrophoresis and transferred to nitrocellulose
membranes The membranes were treated with primary
antibodies overnight at 4°C and incubated with a
HRP-conjugated anti-mouse or anti-rabbit secondary antibody
at room temperature for 1 h The protein bands were
visualized using an enhanced chemiluminescence reagent
(Pierce Biotechnology, USA), according to the
manufac-turer’s instructions
Short interfering RNA (siRNA) transfection
KG1a and Kasumi-1 cells were seeded onto 6-well plates
for 24 h before transfection Control scrambled siRNA
was synthesized and purchased from GenePharma
(Shang-hai Co Ltd., China) SiRNA Bcl-2 (50 nM):
5’-GGGA-GAUAGUGAUGAAG UAUU-3’ [33] or control scramble
sequences were transfected using Lipofectamine 2000
reagent (Invitrogen), according to the manufacturer’s
pro-tocol Briefly, for each well, 5μl Lipofectamine 2000 was
diluted in 250μl Opti-MEM medium (Invitrogen) The
mixture was gently added to a solution containing siRNA
in 250μl Opti-MEMI medium and incubated for 20 min
The mixture was then added to the plates After
transfec-tion with siRNA for 24 h, cells were harvested for further
assay
Statistical analysis Data were presented as mean ± SD One-way ANOVA fol-lowed by Bonferroni multiple comparison was performed
to assess the differences between two groups under multi-ple conditions If the data failed the normality test, the Kruskal-Wallis one-way ANOVA on ranks was used A value of p < 0.05 was considered statistically significant Both Calcusyn software (Biosoft, Ferguson, MO, USA) [34,35] and Jin’s formula [36] were used to evaluate the synergistic effects of drug combinations Jin’s formula is given as: Q = Ea + b/(Ea + Eb-Ea × Eb) Ea+b represents the cell proliferation inhibition rate of the combined drugs, while Ea and Eb represent the rates for each drug respectively A value of Q = 0.85-1.15 indicates a simple additive effect, while Q > 1.15 indicates synergism Combi-nation index (CI) plots were generated using CalcuSyn software A value of CI < 1 indicates synergism
Results
CD34+KG1a and Kasumi-1cells were insensitive to DNR KG1a, Kasumi-1 and U937 AML cells were stained with PE-conjugated CD34 antibody and subjected to flow cytometry to determine the purity of CD34+ cells The percentages of CD34+cells were 99.43 ± 0.60% in KG1a cells, 96.67 ± 0.11% in Kasumi-1 cells, but CD34+ was absent in U937 cells (Figure 1A) After treatment of these three cell lines with different concentrations of DNR for 48 h, MTT and apoptosis analyses showed that DNR inhibited proliferation and induced apoptosis in more mature U937 cells, but not in immature CD34+ KG1a or Kasumi-1 cell lines (Figure 1B, C) This was in accord with previous studies indicating that CD34+ AML cells were insensitive to DNR The concentration
of DNR used in this study was clinically achievable in patients [37,38]
Curcumin suppressed cell growth and induced G1/S cell cycle arrest in both DNR-insensitive and -sensitive AML cell lines
KG1a, Kasumi-1 and U937 cell lines were exposed to curcumin (0-100μM) for 24, 48, 72 and 96 h The cyto-toxic effects of curcumin were determined by MTT assay Curcumin had a significant cytotoxic effect in all tested cell lines in both dose- and time-dependent man-ners (Figure 2B, C, D) The IC50 values at 24, 48, 72, and 96 h were 230.5, 86.9, 60.0, and 35.7μM for KG1a, 68.5, 46.6, 28.8, and 23.5 μM for Kasumi-1, and 58.3, 26.0, 10.6, and 4.4μM for U937 cells, respectively The antiproliferative effects of curcumin in these cell lines were further determined using clonogenic assays Curcu-min inhibited clonogenic growth in a dose-dependent manner, and completely inhibited colony formation at a dose as low as 20μM (Figure S1A, B, Additional file 1)
Trang 5Cell cycle distributions in KG1a, Kasumi-1, and U937
cells were examined after treatment with curcumin for
24 h As shown in Figure 2E, treatment of KG1a cells
with 80μM curcumin resulted in a significant increase
in the percentage of cells in the G1 phase, from 46-62%,
and a decrease in the percentage of cells in the S phase, from 39-23% Similar results were found for Kasumi-1 and U937 cells These results demonstrated that curcu-min induced G1/S arrest in both DNR-insensitive and -sensitive AML cell lines
M1
A
B
99.72 0.18
.*D NDVXPL
X
***
***
.*D NDVXPL
8
***
96.71
Daunorubicin(ȝg/ml)
Daunorubicin(ȝg/ml)
U937
C
Annexin č
PI
0 0.4 0.8 1.6
Daunorubicin(ȝg/ml)
1.65
8.13 0.02
90.19 1.35
10.15 0.00
88.50
4.84
0.89
94.27
0.00 0.37
2.69
26.94
0.00
1.32
4.65 0.01
94.02 0.69
3.64 0.00
95.67 0.42
3.51 0.00
96.07 0.56
3.69
0.05
95.70
14.16
26.56 12.68
46.60 12.39
16.13 48.40
23.08 8.78
19.60 1.62
69.99 0.64
2.75
0.01
96.60
Figure 1 CD34+KG1a and Kasumi-1cells were insensitive to DNR (A) KG1a, Kasumi-1 and U937 cells were stained with PE-conjugated CD34
antibody and subjected to flow cytometry to determine the purity of CD34+cells (B, C) These three cell lines were treated with different
concentrations of DNR for 48 h MTT assay (B) was performed as described in “Methods” and apoptosis (C) was assessed by Annexin V/PI assays.
Cells in the lower right quadrant represent early apoptosis and those in the upper right quadrant represent late apoptosis The graph displays
the means ± SD of three independent experiments * p < 0.05, ** p < 0.01, *** p < 0.001 (compared with control).
Trang 6
K
K
K
K
*0 6
**
Curcumin(ȝM)
K
K
K
K
C
A
Curcumin
Curcumin(ȝM)
E
KG1a Kasumi-1 U937
Curcumin(ȝM)
D
K
K
K
K
B
Curcumin(ȝM)
U937
Figure 2 Curcumin suppressed cell growth and induced G1/S arrest (A) Structure of curcumin (B, C, D) KG1a, Kasumi-1 and U937 cell lines were treated with different concentrations of curcumin for 24, 48, 72, and 96 h MTT assay was performed (E) These three cell lines were treated with different concentrations of curcumin for 24 h and analyzed for DNA content by flow cytometry, as described in “Methods.” The bar represents means ± SD of three independent experiments.
Trang 7Curcumin induced apoptosis through activation of
caspase-3 followed by PARP degradation in both
DNR-insensitive and -sensitive AML cell lines
To determine if growth inhibition induced by curcumin
was a result of apoptosis, the pro-apoptotic effect was
examined using Wright-Giemsa, Hoechst 33342 and
Annexin-V/PI staining Both Wright-Giemsa and
Hoechst 33342 staining showed that curcumin induced
morphological changes such as cell shrinkage and nuclear
condensation, which are typical characteristics of
apopto-sis (Figure 3A; Figure S2A, Additional file 2) These
mor-phological changes were confirmed by flow cytometry
Treatment with curcumin at 40μM for 24 h resulted in
apoptosis rates of 23.5 ± 8.8%, 36.1 ± 5.3%, and 40.1 ±
17.8% in KG1a, Kasumi-1 and U937 cells, respectively
(Figure 3B) Western blotting analysis further showed
that curcumin induced caspase-3 activation and PARP
cleavage, two hallmarks of apoptosis (Figure 3C) Both
Annexin-V/PI and Western blotting showed that
curcu-min induced apoptosis in a dose-dependent manner
U937cells were the most sensitive to curcumin-induced
apoptosis, followed by Kasumi-1, then KG1a cells
Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines
The mechanisms underlying curcumin-induced
apopto-sis were investigated The IAP and Bcl-2 family play an
important role in the regulation of cell apoptosis, and
the effects of curcumin on mRNA levels of c-IAP-1,
XIAP and Bcl-2 were therefore assessed by RT-PCR As
shown in Figure 4A, Bcl-2 mRNA levels were
signifi-cantly down-regulated in both DNR-insensitive AML
cell lines (KG1a and Kasumi-1) and in DNR-sensitive
U937 cells, while the levels of c-IAP-1 and XIAP were
unchanged Western blotting also demonstrated that
curcumin significantly down-regulated Bcl-2 protein
levels in a dose-dependent manner (Figure 4B) These
results suggest that down-regulation of Bcl-2 could
con-tribute to curcumin-induced apoptosis
Disruption of the function of Bcl-2 protein leads to
per-meabilization of the mitochondrial membrane [39] We
therefore investigated the effects of curcumin on MMP
using JC-1 fluorescent dye and flow cytometry Exposure
of the three cell lines to increasing doses of curcumin for
24 h led to a significant reduction in the MMP (Figure
4C) These results suggest that curcumin-induced
apopto-sis is mitochondria-dependent
Curcumin synergistically enhanced the cytotoxic effect of
DNR in DNR-insensitive KG1a and Kasumi-1 cells,
associated with down-regulation of Bcl-2
To determine if curcumin could enhance the cytotoxic
activity of DNR, DNR-insensitive KG1a and Kasumi-1
cells were cultured with combinations of these two drugs at different doses but in a constant ratio (curcu-min to DNR: 20 μM to 0.1 μg/ml, 40 μM to 0.2 μg/ml, and 80 μM to 0.4 μg/ml, respectively) for 48 h, as shown in Figure 5A, B and Table S1 (Additional file 3) Both CalcuSyn software and Jin’s formula were used to determine synergy, and the results were consistent With the exception of co-treatment of KG1a cells with
20μM curcumin and 0.1 μg/ml DNR, which showed an additive effect (CI = 1.03, Q = 0.99), co-treatment with other doses in KG1a cells and with all doses in
Kasumi-1 cells exhibited synergistic effects For example, the combination of 40μM curcumin with 0.2 μg/ml DNR
in KG1a cells caused growth inhibition of 45.12%, com-pared to curcumin (26.31%) or DNR (5.47%) alone, indi-cating synergism (CI = 0.654, Q = 1.49) Notably, co-treatment with 40 μM curcumin and 0.2 μg/ml DNR caused more attenuation of Bcl-2 protein levels than treatment with either agent alone (Figure 5C)
Suppression of Bcl-2 with siRNA induced apoptosis and increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis
To clarify if down-regulation of Bcl-2 by curcumin plays
an important role in this synergistic effect, Bcl-2 expres-sion was suppressed by siRNA and the effect on apopto-sis and DNR sensitivity was examined by flow cytometry Bcl-2 siRNA-induced apoptosis in 24 h (28.58% in KG1a cells, 37.12% in Kasumi-1 cells) was similar to that in cur-cumin-treated KG1a (31.71%, 60 μM, Figure 3B) and Kasumi-1 cells (36.10%, 40μM, Figure 3B), respectively (Figure 6A, B) As shown in Figure 6C, suppression of Bcl-2 by siRNA increased the susceptibility of these cell lines to DNR-induced apoptosis (40.15% in KG1a cells and 86.23% in Kasumi-1 cells), compared to DNR only (3.17% in KG1a cells, 5.94% in Kasumi-1 cells) These results suggest that suppression of Bcl-2 could contribute
to curcumin-induced apoptosis and the synergistic effect
of curcumin and DNR
Curcumin was effective against primary CD34+AML cells The cytotoxic effects of either curcumin and/or DNR on primary CD34+AML cells were also examined CD34+ cells were sorted from BMMCs or PBMCs from 9 AML patients and 8 healthy donors The sorted samples yielded more than 95% CD34+cells with greater than 90% viabi-lity, determined by trypan blue exclusion (Figure 7A) The antiproliferative effects of curcumin on CD34+cells from
3 AML patients (patients 1, 2, 3) and 3 healthy donors (donors 1, 2, 3) were determined by MTT assay, and com-pared with the results of DNR treatment CD34+ cells were treated with curcumin (0, 10, 20, 40, 80μM) or DNR (0, 0.4, 0.8, 1.6μg/ml) for 24 h Curcumin significantly inhibited proliferation of CD34+AML cells, but only
Trang 8
.*D DVXPL
8
Kasumi-1 KG1a
0 40 60 80
A
U937
C
Curcumin(ȝM)
Curcumin(ȝM)
Cleaved caspase-3
0 40 60 80 0 40 60 80 0 40 60 80
KG1a Kasumi-1 U937
Cleaved PARP
GAPDH Curcumin(ȝM) Annexin č
PI
B
Curcumin(ȝM) 0.26
91.83
2.36
5.55
2.99
19.80 1.01
3.83
38.27 0.56
57.34
2.66 0.67
76.79
12.83 0.04 1.88
93.59 4.49
19.96 0.90
25.08 54.06 82.29 12.26
0.43
9.95 0.15 5.30
1.81
7.09
91.09
0.02 7.93
21.09 0.03
70.95
17.22
31.28 0.03
51.47
23.89
34.56 41.55 0.00
Figure 3 Curcumin induced apoptosis through activation of caspase-3 followed by PARP degradation KG1a, Kasumi-1 and U937 cells were incubated with indicated concentrations of curcumin for 24 h (A) Cells were stained with Wright-Giemsa and then examined under a light microscope Arrows indicate apoptotic cells (magnification ×400) (B) Cells were stained with Annexin V/PI to analyze apoptotic cell
populations The graph displays the means ± SD of four independent experiments (C) Western blotting analysis showed cleaved caspase-3 (17,
19 kDa) and cleaved PARP (89 kDa) fragment Three independent experiments were performed with similar results, and representative data are shown.
Trang 9_
0 40 60 80
_
0 40 60 80 KG1a kasumi-1 U937
.*D NDVXPL
8
B
A
0 40 60 80
Bcl-2 c-IAP-1 XIAP ȕ-actin
Red/green ratio (% of
C
Curcumin(ȝM)
Curcumin(ȝM)
GAPDH Bcl-2
0 40 60 80 0 40 60 80
KG1a kasumi-1 U937
Curcumin(ȝM)
0 40 60 80
Green Red
Curcumin(ȝM)
38.27 19.14
80.82 23.86
76.13 37.65
62.33 93.25
6.47
49.91 97.31
50.05 72.29
27.68 92.88
7.08 2.58
0.36
99.63 7.25
92.74 20.25
79.73 84.76
15.22
Figure 4 Curcumin decreased Bcl-2 mRNA and protein levels and caused the loss of MMP KG1a, Kasumi-1 and U937 cells were exposed
to different concentrations of curcumin for 24 h (A) The effects on Bcl-2, c-IAP-1, and XIAP mRNA levels were determined by RT-PCR (B) The effect on Bcl-2 protein levels was determined by Western blotting assay Three independent experiments were performed with similar results, and representative data are shown (C) MMP was estimated by flow cytometry showing decrease in the red to green fluorescence ratio The results shown are representative of three independent experiments The bar represents mean ± SD of three independent experiments.
Trang 10Bcl-2 GAPDH
– + – +
KG1a Kasumi-1
Daunorubicin(0.2ȝg/ml) Curcumin(40ȝM)
C
A
c
a
b
a
KG1a
B
Figure 5 Curcumin synergistically enhanced the cytotoxic effects of DNR associated with down-regulation of Bcl-2 KG1a and Kausumi-1cells were exposed to different concentrations of curcumin, DNR, or their combination as indicated, for 48 h (A, B) CI-effect plots and median-effect plots were generated using CalcuSyn software The points a, b, and c represent CI values for the combinations 20, 40, and 80 μM
curcumin with 0.1, 0.2, and 0.4 μg/ml DNR in a constant ratio, respectively The CI values at ED 50 , ED 75 , ED 90 were 0.667, 0.490, and 0.364 for KG1a cells and 0.529, 0.456, and 0.394 for Kasumi-1 cells, respectively (C) Bcl-2 protein levels were determined by Western blotting assay Three independent experiments were performed with similar results, and representative data are shown.